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NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment

Department of Physiology, 615 Michael St, 605G Whitehead Building, Emory University School of Medicine, Atlanta, GA 30322, USA

View larger version (85K): [in a new window]   Fig. 4. M-C6-NBD-PC is trafficked to the vacuole in an energy-requiring, vesicle-mediated process. (A) The wild-type diploid strain CRY3 was grown to early log phase in SDC at 30°C. Cells were then labeled with DAPI for 10 minutes prior to being placed on ice and chilled. After approximately 10 minutes on ice, cells were labeled with 10 µM DMSO-solubilized lipid for 90 minutes. Cells were then harvested and washed three times with either ice-cold SCNaN3+F or ice-cold SDC. Aliquots from each batch of cells were then incubated in their respective media at 30°C for 1 hour. Cells were subjected to a final wash with ice-cold SCNaN3+F prior to analysis and imaging by fluorescence microscopy. (B) The end4 mutant strain and its isogenic parent strain were grown to early log phase in SDC at 30°C. The isogenic parent and end4 strains were labeled with DMSO-solubilized M-C6-NBD-PC (1.0 µM and 0.5 µM, respectively) on ice for 60 minutes, followed by washing twice in ice-cold SDC. Cells were warmed to 30°C and labeled with FM4-64 (40 µM) for 15 minutes, washed twice in SDC (30°C) and resuspended in SDC (30°C) for 60 minutes. Cells were centrifuged, then resuspended in ice-cold SCNaN3 prior to imaging by fluorescence and differential interference contrast (DIC) microscopy.

View larger version (76K): [in a new window]   Fig. 1. NBD-PC but not NBD-PE is trafficked to the vacuole (v) in yeast. The wild-type diploid strain CRY2 was grown to early log phase in SDC at 30°C. Cells were then labeled with 5 µM DMSO-solubilized lipid for 45 minutes at 30°C. After being washed twice with room temperature SDC, DAPI was added to label nuclear (n) and mitochondrial (m) DNA. Cells were incubated at 30°C for 15 additional minutes, harvested, and washed twice with ice-cold SC-azide prior to analysis and imaging by fluorescence microscopy.

View larger version (93K): [in a new window]   Fig. 2. M-C6-NBD-PC is not trafficked to the vacuole via the endocytic pathway. Strains were grown to early log phase in SDC at 23°C. After a 30 minute incubation at 37°C, cells were labeled for 1 hour with 50 µM of M-C6-NBD-PC-containing vesicles as described in the Materials and Methods. The cells were then washed three times with ice-cold SCNaN3 and harvested prior to imaging by fluorescence microscopy. The cells shown were labeled at the restrictive temperature, 37°C. When labeled at the permissive temperature, 23°C, both strains also exhibited vacuolar labeling (data not shown).

View larger version (79K): [in a new window]   Fig. 3. M-C6-NBD-PC and M-C6-NBD-PE label mitochondria (m) and the nuclear envelope (n) at low temperature. The wild-type diploid strain CRY2 was grown to early log phase in SDC at 30°C. Cells were then labeled with DAPI for 10 minutes prior to being placed on ice and chilled. After approximately 10 minutes on ice, cells were labeled with 5 µM DMSO-solubilized lipid for 90 minutes. Cells were then harvested and washed three times with ice-cold SCNaN3 prior to analysis and imaging by fluorescence microscopy.

View larger version (122K): [in a new window]   Fig. 5. Trafficking of M-C6-NBD-PC to the vacuole requires vesicular transport from the PVC to the vacuole. (A) The sec12-4 mutant strain and its wild-type parent were labeled as described in Fig. 2. (B,C) The wild-type and deletion strains were grown to early log phase in SDC at 30°C and labeled as described in Fig. 1.

View larger version (76K): [in a new window]   Fig. 6. Both monomethyl and dimethyl M-C6-NBD-PE are trafficked to the vacuole in yeast. The wild-type diploid strain CRY3 was grown to early log phase in SDC at 30°C. Cells were then labeled with 5 µM DMSO-solubilized lipid for 45 minutes at 30°C. After being washed twice with room temperature SDC, DAPI was added to label nuclear and mitochondrial DNA. Cells were incubated at 30°C for 15 additional minutes, harvested and washed twice with ice-cold SCNaN3 prior to analysis and imaging by fluorescence microscopy.

View larger version (64K): [in a new window]   Fig. 7. After internalization by flip, M-C6-NBD-PC is sorted to the vacuole and must pass through the PVC. Based on analysis of membrane trafficking mutants deficient in the steps shown in the figure, we determined that M-C6-NBD-PC must pass through the PVC in order to gain access to the lumen of the vacuole. The key result leading to this model is that class E vps mutants, which are defective in vesicular transport from the PVC to the vacuole, do not sort M-C6-NBD-PC to the vacuole. Two models of how M-C6-NBD-PC might gain access to the lumen of organelles in the vps pathway are illustrated — namely, internalization into the PVC by either invagination of the PVC or by a PVC-localized flippase (see Discussion for details).